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Specificity of ribozyme designed for mutated DHFR mRNA
H Kobayashi1, N Kim, M E Halatsch
1Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY 10029.
Biochemical Pharmacology
|April 29, 1994
Summary
Developing antifolate resistance in leukemia cells can lead to drug resistance. A synthesized ribozyme targeting mutated dihydrofolate reductase mRNA unexpectedly cleaved wild-type RNA, highlighting potential risks in targeted therapies.
Area of Science:
- Molecular Biology
- Cancer Research
- Drug Resistance
Background:
- Sequential exposure to trimetrexate (TMQ) and methotrexate (MTX) induced antifolate resistance in MOLT-3 human acute leukemia cells.
- The resistant cell line, MOLT-3/TMQ800-MTX10,000, exhibited two specific point mutations in the dihydrofolate reductase (DHFR) gene.
Purpose of the Study:
- To synthesize a ribozyme targeting the double-mutated DHFR mRNA in resistant leukemia cells.
- To evaluate the specificity and efficacy of the designed ribozyme against mutated and wild-type DHFR RNA.
Main Methods:
- Generation of a drug-resistant leukemia cell line (MOLT-3/TMQ800-MTX10,000) through sequential antifolate treatment.
- Identification of point mutations in the DHFR gene of the resistant cell line.
- Synthesis of a ribozyme designed to specifically bind and cleave the mutated DHFR mRNA.
- In vitro assessment of ribozyme activity against both mutated and wild-type DHFR RNA substrates.
Main Results:
- The synthesized ribozyme effectively cleaved the target double-mutated DHFR mRNA.
- Unexpectedly, the ribozyme also demonstrated significant cleavage activity against the wild-type DHFR RNA substrate.
- This indicates a lack of complete specificity in the ribozyme's action.
Conclusions:
- Targeting mutated mRNA of essential enzymes with ribozymes requires careful consideration due to potential off-target effects.
- The observed cross-reactivity suggests caution when employing ribozymes as specific therapeutic agents against mutated essential genes.
- Further research is needed to enhance ribozyme specificity for safe and effective clinical application in cancer therapy.